Evidence That IAA Conjugates Are Slow-Release Sources of Free IAA in Plant Tissues

Abstract

Evidence that indoleacetic acid (IAA) conjugates are metabolzed via enzyme-catalyzed hydrolysis to free IAA and that their biological activties are related to the rates at which they are hydrolyzed by the tissue is presented. These conclusions are based on the following observations. Slow but continuous decarboxylation of the IAA moiety of IAA-L-lanie and IAA-glycine occurs when these conjugates are appied to pea (Piswm sativwm L. cv. Alaska) stem segments. Inasmuch as IAA conjugates are protected from peroxidase-catalyzed oxidative decarboxylation, the conjugates are probably hydrolyzed and the freed IAA then further metabo-lized. Free IAA and IAA-L-alanlne are converted, by pea stem tissue, Into the same metaboltes. The metabolism Is enzymk, since conjugates of IAA with the D-isomers of the amino acids are inactive. Ethylene production induced by IAA-L-alanine and by IAAglycine is correlated with their hydrolysis, as indicated by their decarboxylation and with the appearance or nonappearance of IAA metabolites In the tissues. Indoleacetylamino acid conjugates support the growth of plant tissue cultures (5, 10) and have a variety of effects on tissue differentiation (7). The activities of the conjugates differ greatly from the activity of free IAA and also differ greatly among themselves, depending on the amino acid moiety. Some of the conjugates, such as IAA-L-alanine, mimic a frequently renewed supply of free IAA (7). These findings suggest that the conjugates are slowly hydrolyzed, giving a constant, steady-state concentration of free IAA. If the slow-release hypothesis for the mode of action of the IAA conjugates is correct, one would expect that the rate of hydrolysis of the conjugates would control the availability of free IAA to the tissues and would, thus, be correlated with biological activity and that the free IAA released upon hydrolysis of the applied conjugates would be metabolized into some of the same products formed when tissues are directly treated with free IAA. These possibilities were tested by studying the metabolism of IAA conjugates synthesized from [1-_4CJIAA. MATERIALS AND METHODS Synthesis of IAA Conjugates. Radioactive IAA-L-alanine and IAA-glycine were synthesized by the mixed anhydride procedure of Wieland and Horlein (12), with the modifications reported by Hangarter et al. (7). The unlabeled IAA conjugates were also synthesized by the mixed anhydride method (7). 'Supported in part by National Science Foundation Grant PCM-76-07581-AOl. Michigan Agricultural Experiment Station Journal Article 9952. 'Fingerprints' of Auxin Metabolites. Pea seeds (Pisum sativum L. cv. Alaska) were sterilized by soaking for 15 min in 0.5% NaOCl solution containing 0.01% sodium laurylsulfate. The seeds were then rinsed several times in sterile distilled H20 and germinated in the dark on water-agar (0.6%). Six to 7 days after planting, the epicotyls were excised aseptically, cutting them just below the plumular hook and 2 cm above the seed. The epicotyls were then cut into 1-cm segments. Approximately 10 g of segments were transferred into 125-ml flasks containing 25 ml of autoclaved Murashige and Skoog (9) nutrient medium and 1.0 ,Ci of [1-14CJ IAA or [l-14C]IAA-amino acid (10 mCi/mmol). The tissue was incubated in the dark at room temperature, with shaking for 72 h. The medium was filtered off on a Buchner funnel, and the tissue was rinsed several times with distilled H20. The treated tissues were then placed in beakers with 95% ethanol (2 ml/g). Unlabeled IAA and appropriate IAA conjugates were added at this time to provide carrier and chemically detectable internal chromatographic standards. The tissues were boiled for 3 min in the ethanol, then homogenized with a mortar and pestle. The homogenate was filtered with suction on a Buchner funnel, and the residue was rinsed several times with 80% ethanol. The combined ethanol fractions were concentrated to a viscous residue by distillation of the solvent at reduced pressure and then resus-pended in 2-propanol:water (1:1, v/v). This solution was passed over a DEAE-Sephadex-acetate column (1 x 10 cm). The column was washed with 25 ml 2-propanol:water (1:1, v/v). The column was then eluted with a linear gradient of 0 to 3% H3PO4 in 2-propanol:water (1:1, v/v). The fraction containing the acidic in-doles (eluting between 20 and 25 ml) was collected, and the propanol was evaporated at reduced pressure. The remaining aqueous fraction was immediately extracted three times with 25 ml diethyl ether. The ether fractions were pooled and washed once with 2 ml water. The ether fraction was then evaporated to dryness at reduced pressure and resuspended in 0.5 ml 95% ethanol. This alcohol solution, containing the acidic, ether-soluble metabolites was used for TLC.